Feed supplement compositions

ABSTRACT

A process for the preparation of thermostable, discrete, solid particles containing lactoferrin embedded in a protein- and/or starch-based carrier matrix by mixing the carrier matrix, lactoferrin and an optional internal lubricant in a horizontal screw extruder, extruding and cutting said mixture into pieces and optionally drying said pieces as well as their use as food or animal feed supplements.

The present invention relates to a process for the preparation ofcompositions with improved properties for use in nutrition, especiallyin animal nutrition and to the compositions thus obtained.

More precisely, the present invention relates to a process for thepreparation of compositions useful as food or feed supplementscomprising lactoferrin and to the compositions thus obtained.

The present invention also relates to the use of compositions comprisinglactoferrin as a food feed supplement, especially for improving thehealth status of animals and to feed compositions comprising asupplement on the basis of lactoferrin.

Lactoferrin is a naturally occurring single chain glycoprotein with amolecular weight of about 80′000 D containing 1-4 glycan units which canbe isolated from milk or milk derivatives of many animals by differentmethods.

U.S. Pat. No. 4,791,193 describes a method for the preparation of bovinelactoferrin in high purity wherein bovine milk is contacted with aweakly acidic cation exchange resin containing carboxymethyl groups andeluting the adsorbed lactoferrin with sodium chloride solutions.

WO 89/04608 discloses a process for extracting pure lactoperoxidase andlactoferrin from milk serum. The milk serum is microfiltered and passedthrough a strong cation exchanger at a high flow rate for selectiveadsorption of lactoferrin and lactoperoxidase and selective andsuccessive elution with saline solutions of different concentrations.

British patent application No. 2179947 discloses the isolation oflactoferrin from whey by ultrafiltration and weak cation exchangechromatography at approximately neutral pH.

WO 93/13676 discloses a process for isolating lactoferrin from milk orwhey by adsorbing it to a cation exchange resin at a high superficialvelocity and at a high liquid load, eluting it with a salt solution,e.g., sodium chloride in phosphate buffer, and, optionally, drying it.

WO 95/22258 reports a method for substantially purifying humanlactoferrin from milk including the milk of a transgenic animalharboring a gene which encodes the expression and secretion of humanlactoferrin in its milk by contacting the milk with a strong cationexchange resin under elevated ionic strength conditions and eluting thehuman lactoferrin with a stepwise or linear salt gradient.

WO 02/096215 describes an industrial process for the isolation of bovinelactoferrin and other proteins from large volumes of, e.g., milk or milkderived products such as whey, using Expanded Bed Adsorption (EBA)technology. The liquid is applied to high density adsorption materialwith a mean particle size of at most 150 μm and a particle density of atleast 1.5 g/ml followed by elution of the protein from the adsorbent.

Lactoferrin has iron-binding properties, exhibits antimicrobial andantiviral activity and shows immunomodulatory functions in thehost-defense system.

Lactoferrin has therefore been used as feed additive to increase thegrowth and health status of animals.

U.S. Pat. No. 4,726,948 discloses anti-bacterial compositions comprisinglactoferrin and lactoperoxidase for the treatment of gastrointestinalinfections as components of foodstuff and animal feedstuff.

U.S. Pat. No. 4,977,137 discloses lactoferrin from non-processed milk asa dietary ingredient which promotes growth of the gastrointestinal tractof human infants and infant non-human animals.

U.S. Pat. No. 5,656,591 discloses the use of antimicrobially activepeptides derived from lactoferrins in combination with an antibiotic forthe treatment of foods and non-medical products.

It is an object of the present invention to provide lactoferrincontaining granules with improved product properties suitable fornutrition, especially for animal nutrition, and a process for themanufacture thereof. The invention also relates to animal feed enrichedwith the improved lactoferrin containing granules.

While different lactoferrin species may exist which differ in the numberand sequence of amino acids as well in the glycan moiety, the term“lactoferrin” as used herein below comprises all these differentmolecules irrespective of their origin, whether natural or synthetic,prepared by purification from a natural source, by classical chemicalsynthesis or by recombinant methods, as long as the molecule has ahomology of at least 60% in amino acid sequence with a naturallyoccurring lactoferrin molecule and exhibits at least one of itsbiological activities, especially its antimicrobial activity. The term“sequence homology” or “homology in sequence” relates to amino acidsequences which can be longer or shorter than that of a naturallyoccurring molecule with one or multiple exchanges, insertions and/ordeletions of one or more amino acids. Homology of at least 60% includeshomologies of 70, 75, 80, 85, 90, 95, or higher percentages. The term“lactoferrin” includes lactoferrin on the market; lactoferrin isolatedfrom mammalian (humans, cows, sheep, goats, horses, camels, etc.) milk,such as colostrum, transitional milk, matured milk, milk in laterlactation, or from processed products thereof, such as skim milk,evaporated skim milk, butter milk, milk protein concentrate, whey,cheese whey, casein whey, desalted whey or whey concentrate. Thelactoferrin of preferred interest is bovine lactoferrin. It is producedin large amounts from whey which is a by-product from the cheesemanufacturing process. Different processes are known and used in theproduction of bovine lactoferrin comprising different chromatographicprocedures and including a final drying step of the purified andconcentrated lactoferrin-containing solution. Non-formulated lactoferrinthus obtained, viz. after drying by a conventional drying process, suchas freeze drying, spray drying, or vacuum drying, in more or less dryform and free-flowing, however, in the form of a fine powder, is thenblended with the feedstuff or with a carrier material to yield first apremix which is useful as feed additive. During that multi-stepprocedure which is complex, denaturation and loss of biological activityoccurs on each stage and the compositions obtained, containing thelactoferrin in highly dispersed form, are further subject to degradationand do not possess a high shelf-stability.

The present invention overcomes this disadvantage and providesspecifically formulated lactoferrin compositions which are shelf-stable,in the form of discrete, solid particles which contain lactoferrinembedded in a particular carrier matrix.

WO 98/18610 discloses a multistep method for continuously encapsulatingor embedding a component in a matrix comprising admixing a compound witha cooled plasticized matrix material in the presence of a plasticizerunder low shearing conditions, extruding said formable mixture through adie to obtain an extrudate and cutting said extrudate into pieces. Thespecification mentions more than eight hundred compounds specificallywhich can be embedded in such a plasticized matrix material in thepresence of a plasticizer and shows in FIG. 1 a schematic representationof the process using a commercially available extruder. FIGS. 2 and 3show schematically extrusion processes, exemplary extruder barrel andscrew configurations.

The present invention uses a similar technology as described in WO98/18610 for the preparation of thermostable, discrete, solid particlescontaining lactoferrin embedded in a protein- and/or starch-basedcarrier matrix. The process of the present invention is characterized bythe steps of

-   -   (a) adding to a horizontal screw extruder the carrier matrix,        lactoferrin and, optionally, an internal lubricant,    -   (b) mixing these components in the extruder under low shear and        temperature conditions to obtain a homogeneous formable mixture,    -   (c) extruding and cutting said formable mixture-into pieces and,        optionally,    -   (d) drying said pieces.

The invention also relates to the use of the above defined particles,especially when prepared according to the above-defined process, as foodor animal feed supplements, to such supplement compositions themselvesand finally to food or animal feed supplemented with such a supplementcomposition.

In accordance with the process of the present invention the discrete,solid particles or granules can be prepared in any horizontal screwextruder via pasta extrusion using an appropriate extrusion screwconfiguration for achieving low shear and low temperature mixing. Forexample, a combination of alternating small pitch conveying elementswith distributive mixing elements, that are staggered at an angle toeach other for providing axially oriented leakage flow inside theextruder barrel may be employed. The combination of alternatingconveying elements with distributive mixing and kneading elements causethe material flow to be continuously interrupted without shearing of themass thus resulting in mixing of the material at low mechanical energyinput. Co-rotating intermeshing twin screw extruders, such as thoseavailable from Buhler, Switzerland; Clextral, France; Werner andPfleiderer, Germany, APV, England or Wenger, USA; or a Co-Kneader,available from Buss, Switzerland, are preferred, since they providesuperior mixing action compared to other single screw extruders.

The carrier matrix consists of edible components, well-tolerated byanimals. The main components are protein containing ingredients and alltypes of starches, naturally occurring, in purified and/or chemicallymodified form or as constituents of ground cereals, such as cerealflours or grits, preferably a cereal or cereal byproduct. In a preferredembodiment mere starch-based mixtures, especially mixtures of wheatstarch and durum wheat flour (semolina) are used.

Lactoferrin is added to the carrier matrix either in form of a solution(diluted or concentrated) or as a powder (more or less dry), if desiredin form of a premix.

To the carrier matrix there may be added, if desired, agents influencingthe hydrophobicity or the water-binding capacity of the matrix. Theaddition of hydrophobic compounds helps to prevent or delay penetrationof water or gastric juice into the matrix thereby delaying the releaseof the embedded lactoferrin. Compounds with high water binding capacitymay bind water which penetrates the particles or prevent water fromdissolving the matrix thereby also preventing or delaying the release oflactoferrin from the carrier matrix. The additional compounds used tocontrol the rate of release of lactoferrin may be used in amounts of upto 30% (w/v), preferably from about 5-15% (w/w).

To the carrier matrix may also be added vegetable oils or fats as aninternal lubricant for the mixing and extrusion process and to avoid theformation of dust. This is particularly desirable when lactoferrin isadded to the matrix in form of a dry powder. Any commercially availablevegetable oil may be used and added up to about 10% (w/w), preferably upto about 5% (w/w) of the final weight of the solid particles. Aparticularly preferred oil is refined soy oil.

Finally, physiologically active and compatible minerals, such asmagnesium, zinc or sodium salts, e.g., sulfates, and salts having animpact on the pH value, e.g., acetates, citrates or phosphates may beadded to the carrier matrix in amounts to be adapted to the actualneeds.

The proportion (w/w) of the carrier matrix in the feed supplementcomposition is at least 50%, e.g., from about 60-95% and preferablyabout 75%.

The components of the carrier matrix can be blended and introduced intothe screw extruder as a premix or can be added individually. Lactoferrincan also be added already to the premix as a dry powder and beintroduced into the extruder together with the carrier matrix. However,it is preferred to add lactoferrin as a solution separately to moistenthe mixture. Alternatively, an oil component is added separately asinternal lubricant. During homogenizing and extrusion the extruder iscooled to secure a temperature in the extruder and of the mixture, viz.under working conditions, of below 60° C., normally of from 20-50° C.,preferably 35-40° C.

The lactoferrin to be used in the present process is commerciallyavailable lactoferrin or can be prepared in accordance with methodsdescribed in the state of the art. The preferred source is bovine milkor whey. The purity of the lactoferrin is not essential but should be atleast 80%, preferably greater than 90% and in case of freeze driedmaterial is normally 90-95%.

In the process of the present invention lactoferrin is added to theextruder preferably in form of a solution since this is the form inwhich it is obtained in most of the processes of its production frommilk or whey, viz., by elution from an absorption column. The preferredtechnology for obtaining purified lactoferrin from milk or whey is theEBA technology, as described, e.g., in WO 02/096215, WO 01/85329 or WO99/65586, contents of which is herewith explicitly incorporated into thepresent specification.

The lactoferrin content of the granules into which lactoferrin isembedded is from about 6% to about 50% (w/w) and in specific embodimentspreferably 8-12% (w/w) or about 20-30% (w/w).

The homogenized mixture is extruded through a die plate and the extrudedpasta strands are cut into distinct spherical particles.

The shape and dimensions of these granules of the present invention aredependent mainly on the form and diameters of the extrusion dies, thespeed by which the mixture is extruded and the speed of the cutter andto a lesser extent on the conditions of a subsequent drying process.Convenient extrusion dies may have aperture diameters of 0.1-5 mm,preferably 0.5-1 mm. About 85% of the resulting particles have a size of0.2-0.8 mm, preferably 0.3-0.6 mm.

The extrudate rope may be cut at the die face using a rotating cutter,pelletizer or rotating knives. In other embodiments, the extrudate ropemay be cut away from the die using conventional cutting or forming meansfor producing pellets or tablets. The cut pieces may have a length:diameter ratio (l/d ratio) of about 0.5 to 10, preferably about 1.Depending upon their moisture the granules are more or less dust-freeand edgy.

In accordance with the process of the present invention, the particlesize may be varied to control the surface to volume ratio of the pelletsor granules for achieving a desired controlled release of thelactoferrin. The particle size may be varied, for example, by the use ofdifferent diameters for the extrusion die openings. Particle size mayalso be varied by the use of a variable speed cutter either at the dieplate at the end of the extruder or away from the extruder after theropes have been conveyed for a short distance. By varying the speed ofthe cutter, the size of the cut pieces may be varied for a givenextruder throughput. The use of a variable cutter which is spaced ashort distance from the die plate, for example, between about 0.5 metersto about 5 meters permits further surface cooling, further surfacedrying, and reduces stickiness to provide better cutting of the ropesinto pellets.

After cutting, the resulting pieces or granules may be dried to asufficiently low moisture content which assures a sufficiently prolongedstorage stability of shelf life. For example, the pellets may be driedto achieve a storage stability or shelf life of at least about ninemonths, preferably at least about eighteen months.

The drying may be performed using conventional drying equipment usingdrying temperatures which do not adversely affect the stability oflactoferrin. Exemplary drying temperatures may range from about 10° C.to about 90° C., preferably from about 200° C. to about 60° C. Thedrying may be conducted to achieve a moisture content of less than about30% by weight, preferably less than about 12% weight, for example, fromabout 6% to about 9% by weight. A preferred drying method uses afluid-bed dryer. After drying the particles may be less edgy due tomutual abrasion. If desired, the dried particles may be coated withfilm-building substances to provide them with specific desirableattributes using conventional coating apparatus and methods.

The granules into which lactoferrin is embedded in accordance with thepresent invention are useful as food or feed additives and can be mixedor processed with conventional and specific feed for all kinds ofanimals including humans, e.g., pigs, ruminants, poultry, fish, pets oranimals to be found in zoological gardens, in amounts to provide adesired daily uptake, normally of about 0.5 to about 200 mglactoferrin/kg body weight of animal depending upon its type, age and/orhealth state. If desired the feed supplemented with the lactoferringranules is pelleted using a conventional type of pellet press. It isobvious to those skilled in the art that the particles of the presentinvention although mainly useful as supplements for animal feed can alsobe used for the production of other formulations, e.g., cosmetic orpharmaceutical preparations.

Because of their favorable particle size and the fact that thelactoferrin is embedded in a stabilizing matrix the granules of thepresent invention have improved properties, especially as feed additiveswhen compared with non-embedded lactoferrin.

Lactoferrin embedded into a granulated carrier matrix in accordance withthe present invention does not only exhibit an improved stability andlonger shelf-live and is more slowly released in the gastrointestinaltract when compared with non-embedded freeze-dried material butsurprisingly increases gastrointestinal absorption of amino acids, thefeed conversion rate (FCR) and the daily weight gain of the animal.These effects have been found, e.g., in anastomosed pigs and weanerpiglets, respectively.

The solid lactoferrin-containing particles in a thermostable form of thepresent invention do not only provide resistance against negativeinfluences of elevated temperatures and prolonged time of storage butalso against higher temperatures, e.g. above 80° C., to which feed isexposed during processing.

The invention is further illustrated by the following examples.

EXAMPLE I

To a double-screw extruder type WP 58 Continua 22.5 kg of wheat starch,12.5 kg of durum wheat flour and 12.5 kg Lactoferrin were added as apremix. Water to moisten the mixture and 2.5 kg of soy oil (refined)were added separately. During homogenizing and extrusion the extruderwas cooled and kept at a temperature not exceeding 35° C. usingsaturated brine.

The homogenized mixture was extruded through a pelletizing die plate(diameter of dies: 0.5 mm) and the extruded pasta strands were cut atthe die plate into pieces of about 0.5 mm length using a rotatingcutter. After extrusion the lactoferrin containing granules were driedusing a fluid bed dryer at an intake air temperature of max. 50° C.

The following composition was used to prepare granules useful as animalfeed additive: % (w/w) Lactoferrin 25 Wheat starch 45 Durum wheat flour(semolina) 25 Soy oil (refined) 5

In further embodiments compositions with different weight ratios of theabove components were used under the same process conditions: % (w/w)Lactoferrin 12 25 50 Wheat starch 40 40  3 Durum wheat flour 43 15 42Wheat gluten — 12 — Soy oil  5  8 — Peanut oil, hardened — —  5

EXAMPLE II

To a double-screw extruder type WP 58 Continua 20.0 kg of wheat starch,24.5 kg of durum wheat flour were added as a premix. An enrichedlactoferrin solution obtained via chromatography of whey concentrated toabout 25% protein content (of which about 90% was LF) was added. 2.5 kgof soy oil (refined) were added separately. During homogenizing andextrusion the extruder was cooled and kept at a temperature notexceeding 35° C. using saturated brine.

The following composition was used to prepare granules useful as animalfeed additive: % (w/w) Lactoferrin (dry matter) 6 Wheat starch 40 Durumwheat flour (semolina) 49 Soy oil (refined) 5

In further embodiments compositions with different weight ratios, addingan enriched lactoferrin solution with about 30% protein content (ofwhich about 90% was LF) were used under the same process conditions: %(w/w) Lactoferrin (dry matter) 6 8 10 Wheat starch 41.5 39.5 37.5 Durumwheat flour (semolina) 47.5 47.5 47.5 Soy oil (refined) 5 5 5

1. A process for the preparation of thermostable, discrete, solidparticles containing lactoferrin embedded in a protein-and/orstarch-based carrier matrix which process is characterized by the stepsof (a) adding to a horizontal screw extruder the carrier matrix,lactoferrin and, optionally, an internal lubricant, (b) mixing thesecomponents in the extruder under low shear and temperature conditions toobtain a homogeneous formable mixture, (c) extruding and cutting saidformable mixture into pieces and, optionally, (d) drying said pieces. 2.A process as claimed in claim 1 wherein the carrier matrix compriseswheat starch and durum wheat flour.
 3. A process as claimed in claim 1wherein lactoferrin is added in form of an aqueous solution or a drypowder.
 4. A process as claimed in claim 1 wherein a vegetable oil isused as internal lubricant.
 5. A process as claimed in claim 1 whereinthe temperature in the extruder under working conditions is below about60° C.
 6. Thermostable, discrete, solid particles containing lactoferrinembedded in a protein- and/or starch-based carrier matrix. 7.Thermostable, discrete, solid particles containing lactoferrin embeddedin a protein- and/or starch-based carrier matrix obtained by a processas claimed in claim
 1. 8. Solid particles according to claim 6 for useas animal feed or food supplements.
 9. A food or animal feed supplementcomposition on the basis of solid particles according to claim
 6. 10. Afood or an animal feed supplemented with a composition as claimed inclaim 9.